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. Author manuscript; available in PMC: 2014 Feb 22.
Published in final edited form as: J Mol Biol. 2012 Nov 15;425(4):725–737. doi: 10.1016/j.jmb.2012.11.012

Figure 4. Identification of FBF-2 amino acids that are necessary for the interaction with CPB-1.

Figure 4

(A) Single alanine FBF mutants were tested for biding to CPB-1 with the NiNTA pulldown assay. The first two panels show the controls with CPB-1 or FBF only, mutation of FBF Tyr 479 to Ala completely disrupted the interaction with CPB-1 (forth panel). (B) The affinity of fl-CPB-1 for FBF wild-type and FBF alanine mutants was determined by fluorescence polarization assays. Fluorescence polarization is plotted as a function of FBF concentration and fitted to the Hill equation to determine the apparent equilibrium dissociation constant Kd,(shown in the table; the Hill coefficient was set to 1). The significance (* P-value <0.05) was determined by Student-t-test versus wild type FBF. A graphical summary of the effect of single alanine mutations in the Y479-T485 loop is shown. (C) FBF residues that affect binding to CPB-1 are highlighted in the crystal structure of FBF-2 in complex with PME RNA29. The 8 PUF repeats (R1–R8) are colored blue and grey, the loop connecting repeats seven and eight encompassing residues 479–485 is in red, key residues for the interaction with CPB-1 are shown as spheres.